An investigation of the organization and biogenesis of endoplasmic reticulum (ER) membranes is proposed using rough microsomes (RM) derived from rat liver. Specific proteins (ribophorins, cytochrome P-450, esterases, etc.) will be isolated and characterized as to their peptide composition, the exposure of functional sequences on either membrane face and functional modifications such as phosphorylation and glycosidation. Two membrane proteins (ribophorins), have been discovered, which are characteristic of rough portions of the endoplasmic reticulum. Their structure and organization within the membrane will be further studied. Their involvement in ribosome binding and possible participation in the vactorial discharge and processing of nascent chains will be investigated. The spatial arrangement of membrane proteins will be correlated with mechanisms of their insertion into the ER membrane. Using antibodies against specific membrane proteins we will recognize products of in vitro translation which may be synthesized as precursors. These may contain extra polypeptide sequences which, by analogy with those in secretory proteins, may serve to determine the fate of the polypeptides and be cleaved by membrane proteases. The localization, characterization and isolation of proteases(s) responsible for cleavage of precursor peptides of secretory and membrane proteins will be undertaken. The reconstitution of functional ER membranes will be attempted using purified proteins and microsomal lipids to elucidate intrinsic molecular interactions which determine the spatial organization of membrane components.